Juvenile hormone: Its assay and effects on pupae of Manduca sexta

Non-diapausing pupae of Manduca sexta were used to develop a bioassay for juvenile hormone (JH). The period of maximal sensitivity to Cecropia C17-JH injected in olive oil was found to be 24 to 30 hr after pupal ecdysis at the time of the beginning of epidermal retraction. The dose-response curves for C16-, C17-, and C18-JH at 29±1 hr after pupal ecdysis were determined and those for the latter two compounds were found to be similar and to be linear between 10^?2 and 10 μg/g body weight. C16-JH was 300 times less active than C17- and C18-JH in this bioassay. The effectiveness of 0·5 μg/g C18-JH at 29±1 hr was determined by the carrier media in which it was injected. The highest scores were obtained when the carrier was light mineral oil or loive oil whereas the lowest scores were obtained using 10% BSA or Tween 80. These scores are consistent with the kinetics of equilibration of the injected JH with the haemolymph. Thus, the injected hormone is more effective when it slowly leaks into the blood, presumably because it is metabolized much more slowly.     

Assays on Rhodnius for juvenile hormone activity

Contrary to expectation the effect of a given quantity of juvenile hormone (JH) topically applied to the abdomen of Rhodnius is greatly reduced if the cuticle is thin; either newly stretched by a large meal or wasted by prolonged starvation. The active substance apparently enters the body too rapidly and is eliminated in metabolism.The activity of the juvenile hormone and the most active JH-mimics is increased ten to fifteenfold if they are topically applied in dilute solution in peanut oil or triolein.Assays of the juvenile hormones of Cecropia at a concentration of 1 : 1000 in peanut oil (suitably diluted with octane) show that synthetic (racemic) C₁₈-JH of Röller (JH-1) is considerably less active than the natural enantiomorph; and the synthetic C₁₇-JH of Meyer (JH-2) has only about two-thirds of the activity of JH-1. Using this method of application, the natural juvenile hormone applied to the fifth instar larva of Rhodnius will cause a 50 per cent suppression of adult characters (grade 10 in the assay) at a dose of about 15 ng.     

Excretion of juvenile hormone and its metabolites in the locust, Locusta migratoria

Racemic synthetic ³HC₁₈ juvenile hormone, dissolved in paraffin oil, was injected into adult Locusta migratoria and the excreted radioactive material in the faeces was determined. Within 48 hr two-thirds of the injected radioactivity can be recovered in the frass, half of it within 3 hr. The remaining one-third of the injected label is incorporated or is released as water. Adult locusts of either sex or of different ages show no difference in the metabolic pathways of the JH and its excretion rate.The excreta contain as a degradation product 7-ethyl-3,11-dimethyl-cis-10,11-epoxy-trans, trans-2,6 trideca-dienoic acid, the corresponding dioldienoic acid and the dioldienoic methyl ester. Unchanged Cecropia JH was also found in the frass. The radioactive hormone, as well as the metabolites, were excreted mainly by the Malpighian tubules; smaller amounts of the radioactive material were also found in the fore-, mid, and hindgut.     

Changes in the morphogenetic response of Tenebrio molitor pupae to juvenile hormone in relation to age

Morphogenetic effect of juvenile hormone (JH) and its analogues, dodecyl methyl ether, ethyl trimethyl dodecadienoate and methylenedioxyphenoxy-6-epoxy-3-ethyl-7-methyl-2-nonene, on carefully timed Tenebrio pupae was determined. These results show that the response of pupal epidermal cells to JH varied with age during the first 48 hr after larval-pupal ecdysis. The pupae showed low morphogenetic response soon after pupal ecdysis but their response increased gradually until 18 hr. The response to JH decreased in pupae older than about 32 hr; and 48 hr old pupae were unresponsive to low doses of JH employed in this study. Age-related differences in the pattern of response of the individual body regions to JH were also observed.The synergistic effect of 1 μg of ecdysterone with these JH compounds was also tested in relation to the age of Tenebrio pupa. The results show that the synergistic effect of ecdysterone was generally limited to >18 hr old pupae. This suggests that the physiological basis of the synergistic effect of ecdysterone may be the latter's ability to synchronize epidermal cells.The significance of these observations in the analysis of time of action of juvenile hormone is discussed.     

Activité comparée des hormones juvéniles en C-18 (JH-I) et en C-16 (JH-III) chez Locusta migratoria

The comparative activity of C-16 and C-18 juvenile hormones is studied in Locusta migratoria on four well-known physiological functions of the corpora allata by means of a single injection of a solution of hormone in oil at doses of 50, 100, and 200 μg/animal. Judged on morphogenesis and pigmentation, JH-I (C-18 JH) as well as JH-III (C-16 JH) show a real juvenilizing effect. The potency of JH-I is much higher than that of JH-III because the first hormone only produces supernumerary larvae and most modified green animals. JH-I counterbalances exactly the lack of CA on the gonadotropic function whereas JH-III allows only about 50 per cent development of oöcytes. The cardiotropic activity of JH-I is similar to that of the CA. The C-18 juvenile hormone is until now the only studied ‘juvenilizing’ compound which increases the heartbeat. JH-III appears to have no noticeable effect on the heart.These results combine to prove that only JH-I has an activity similar to the Locusta corpora allata on morphogenesis, pigmentation, ovarian maturation, and the cardiac activity of L. migratoria.     

Synthesis and bioassay of isoprenoid 3-alkylthio-1,1,1-trifluoro-2-propanones: Potent,selective inhibitors of juvenile hormone esterase

Four 3-alkylthio-1,1,1-trifluoro-2-propanones with juvenile hormone-like side chains were prepared from citronellol and homogeraniol. These substrates were designed as possible transition-state analogs for the juvenile hormone (JH)-specific esterases present in insects. These four isoprenoid trifluoromethyl ketones were assayed in vitro with JH esterase and general esterases from larvae of the cabbage looper, Trichoplusia ni (Lepidoptera, Noctuidae), and with eel acetylcholinesterase and bovine chymotrypsin. JH esterase inhibition I₅₀ values were in the nanomolar range for all four compounds, while the other esterases had I₅₀'S which were 10³ to 10⁵ higher. The high selectivity of these inhibitors is believed to be due to their similarity in size and functionality to natural JH III. Treatment of T. ni larvae in vivo with solutions of the most active analog, 3-[(E)-4,8-dimethyl-3,7-nonadienylthio]-1,1,1-trifluoro-2-propanone (DNTFP) causes a dose-dependent delay in pupation and a concurrent selective inhibition of JH esterase. These data support the hypothesis that the reduction in in vivo JH titer in larval T. ni is due, in part, to hydrolysis of the hormone by selective esterases. DNTFP appears to be competing with JH for the active site of JH esterase.     

Development and application of a radioimmunoassay for the juvenile hormones

A radioimmunochemical method for the quantification of juvenile hormones from hemolymph and whole body extracts is described. Rabbit polyclonal antiserum developed against a JH III-bovine thyroglobulin conjugate displayed minimal cross-reactivity with juvenile hormone metabolites including juvenile hormone acids, juvenile hormone diols and analogs but substantial cross-reactivity between juvenile hormone homologs. Minimum sensitivity of the assay toward racemic juvenile hormone III was 65 pg. The degree and relative order of cross-reactivities for juvenile hormones I, II and III varied according to the identity of the radioligand used. A method for isolating juvenile hormones from whole body and hemolymph for radioimmunoassay was developed utilizing organic solvent extraction followed by thin-layer chromatography. Noninterfering dyes were used to bracket the position of the hormone on thin-layer chromatography plates. Hemolymph extracts known to contain no JH did not interfere with the assay when this procedure was employed. Radioimmunoassay analysis of hemolymph samples containing known amounts of JH and corrected for recovery yielded the expected results. Quantification of total juvenile hormone in whole body and hemolymph extracts of Manduca sexta was in good agreement with total mass of JH determined by a GC/MS method.     

Studies on the de novo synthesis of juvenile hormone III and methyl farnesoate by isolated corpora allata of adult female Gryllus bimaculatus

Activity of the corpora allata (CA) in vitro of adult female Gryllus bimaculatus was studied following incorporation of radioactivity from [2-¹⁴C]acetate and l-[methyl-³H]methionine into juvenile hormone III (JH III) and its immediate precursor methyl farnesoate (MF). Spontaneously active glands from females reared at 27°C utilized exogenous labelled acetate extensively for synthesis of MF and JH III (incorporation 80–84% at 2 mM acetate). 10⁻⁷ to 10⁻⁵ M exogenous JH III in the incubation medium had no effect on the rate of JH biosynthesis in spontaneously active glands. At 10⁻⁴ M JH III incorporation of acetate into JH III was reduced. The amount of MF was also lowered. JH III treatment (10⁻⁸–10⁻⁶ M) of spontaneously inactive glands led to an increase in the amount of MF. This increase was due to a de novo synthesis. Exogenous farnesol (20–200 μM) increased JH III biosynthesis and the amount of MF, but suppressed [2-¹⁴C]acetate incorporation. Dilution of the endogenous precursors is probably the most important cause of this suppression. As shown by the abnormally high MF levels in farnesol treated glands, epoxidation seems to be a rate-limiting step under certain experimental conditions.     

Larval esterases of the southwestern corn borer, Diatraea grandiosella: Temporal changes and specificity

Disc electrophoresis was used to examine and characterize the esterases present in the fat body, haemolymph, and midgut of last stage larvae of the southwestern corn borer, Diatraea grandiosella. Significant temporal changes were observed in the pattern of the 4 major esterases of the fat body and 3 major esterases of the haemolymph. These changing profiles presumably relate, in part, to a requirement for the degradation of juvenile hormone (JH) in preparation for metamorphosis.The binding capacity of esterases present in the larval midgut towards JH I and three JH mimics (alkyl-3,7,11-trimethyl-2,4-dodecadienoates) was also examined. The midgut of last stage nondiapausing larvae was shown to contain a carboxylesterase which bound all three JH mimics. Another esterase which bound JH I, but not the mimics, was also present. An esterase with a similar electrophoretic mobility was detected in the haemolymph and integument. Since the JH I binding esterase did not bind the JH mimics, the mimics do not appear to synergize JH by inhibiting its ester hydrolysis.     

Morphogenetic effects of juvenile hormone and juvenile hormone mimics on adult development of Drosophila

The morphogenetic effects of t,t-farnesol, Law-Williams juvenile hormone analogue, dichlorofarnesenic acid ethyl ester (DFAEE), and a syntetic racemic or isomeric mixture of C₁₈ juvenile hormone (JH), when applied topically to pharate pupae and adults of D. melanogaster have been studied. Of these various agents tested, only DFAEE and JH affected adult development and eclosion and the pharate pupae were the most sensitive to these agents. The racemic mixture of JH induced the secretion, in the abdomen, of a supernumerary cuticle indistinguishable from that of the pupa; it, in addition, retarded the synthesis of brown eye pigments, general body pigmentation, and affected the differentiation of various internal organs and cuticular structures of the abdomen. By comparing the effects of JH with those of Minute (M) and bobbed (bb) mutations on the adult development, it is suggested that JH, by retarding genetic translation mimics M or bb.     

Comparative metabolism of the Cecropia juvenile hormone

The in vivo and in vitro metabolism of ³H-Cecropia C18-juvenile hormone (JH) was studied in representative species of eight orders of insects. In all orders the major metabolites were found to be the JH-acid, the JH acid-diol, and conjugated polar metabolites thought to be glucosides or glucuronides. The JH-diol was also present in both Tenebrio and Saturniid pupae. In vitro studies revealed two additional metabolites produced by tissue homogenates in the presence of NADPH. On the basis of chromatographic evidence these are tentatively identified as the JH-tetrol metabolite in Cecropia, Thermobia, and Drosophila, and the JH-bisepoxide in Drosophila.     

Inhibition of the larval ecdysis and emergence behavior of the parasitoid Cotesia congregata by methoprene

Parasitism of the tobacco hornworm, Manducasexta, by the braconid wasp Cotesiacongregata, induces developmental arrest of the host in the larval stage. During the final instar of the host, its juvenile hormone (JH) titer is elevated, preventing host metamorphosis. This study investigated the effects of hormonal manipulation of the host on the parasitoid’s emergence behavior. The second larval ecdysis of the wasps coincides with their emergence from the host, and application of the juvenile hormone analogue methoprene to day 4 fifth instar hosts either delayed or totally suppressed the subsequent emergence of the wasps. Effects of methoprene were dose-dependent and no parasitoids emerged following treatment of host larvae with doses >50 μg. Parasitoids which failed to emerge eventually succumbed as unecydsed pharate third instar larvae in the hemocoel of the host. Effects of host methoprene treatment on parasitoid metamorphosis were also assessed, and metamorphic disruption occurred at much lower dosages compared with doses necessary to suppress parasitoid emergence behavior. The inhibitory effect of methoprene on parasitoid emergence behavior appears to be mediated by effects of this hormone on the synthesis or release of ecdysis-triggering hormone (ETH) in the parasitoid, the proximate endocrine cue which triggers ecdysis behavior in free-living insects. ETH accumulated in the epitracheal Inka cells of parasitoids developing in methoprene-treated hosts, suggestive of a lack of hormone release. Thus, the hormonal modulation of parasitoid emergence behavior appears to be complex, involving a suite of hormones including JH, ecdysteroid, and peptide hormones.     

Dependence of juvenile hormone activity on haemolymph factor in Tenebrio molitor pupae

Morphogenetic response of Tenebrio molitor pupae to juvenile hormone (JH) varies with age, it being low during the first 6 hr after pupal ecdysis and reaching a peak at about 18 hr after ecdysis. Parabiosis of 6-hr pupa to a 18-hr-old pupa causes a higher morphogenetic response in 6-hr pupa to JH. A similar increase in morphogenetic response was not noticeable when a 0·45 μm Millipore filter was placed between the parabiotic partners, suggesting that the factor(s) may be macromolecular. This view is further supported by the observation that the Millipore filter did not prevent transport of ecdysterone another agent that enhances morphogenetic response of pupae to JH.     

Juvenile hormone and photoperiodically controlled polymorphism in Aphis fabae: Prenatal effects on presumptive oviparae

All three naturally occurring juvenile hormones (JH's) were shown to have effects on the parthenogenetic/gamic polymorphism of Aphis fabae; they mimicked long day conditions by inducing parthenogenetic forms. When topically applied to fourth instar gynoparae, JH caused the appearance of oviparous/viviparous intermediate morphs in the progeny. JH induced both wing development and embryogenesis in embryonic, presumptive oviparae. Embryogenesis was induced by lower doses of JH. Adult, embryo-containing alatae produced by treatment with high JH doses were capable of flight, and whilst reluctant to reproduce, their few viable progeny were oviparae. They did, however, differ from normal gynoparae in size, occasional presence of scent plaques on the metathoracic tibiae, numbers of secondary rhinaria on the antennae and morphogenetic response to postnatal rearing in long day conditions. The presumptive, oviparous embryos most sensitive to JH treatment were shown to be ca 323 μm in length, close to the stage where their germaria differentiate as parthenogenetic or gamic. Similar effects were observed in the progeny of JH-treated, teneral adult gynoparae but there was no effect on the morph of progeny of long day, alate virginoparae. The JH's differed in potency in the order JH I > JH II > JH III. The treatment of fourth instar gynoparae also induced a terminal batch of apparently normal viviparous progeny in a number of aphids. This result was obtained even at JH doses below threshold for the appearance of oviparous/viviparous intermorphs.     

Juvenile hormone induction of pheromone gland PBAN-responsiveness in Helicoverpa armigera females

The maturation of corpora allata (CA) and the competence of pheromone glands in the adult moth Helicoverpa armigera, are both age-related and appear to be correlated. Sex pheromone glands of pharate adults do not produce sex pheromone independently, nor do they respond to exogenous PBAN. Newly emerged moths produce significantly less pheromone than day one moths. JH (juvenile hormone) II was found to be the main JH form produced by CA in vitro. JH II primed pheromone glands of pharate adults to respond to PBAN. In addition, injection or topical application of JH II to newly-emerged females induced pheromone production in the presence of PBAN. Our findings suggest that JH is involved in the initiation of pheromone production of Helicoverpa armigera.     

Activities of hormone metabolizing enzymes in house flies treated with some substituted urea growth regulators.

The insect growth regulators (IGR) TH 6038 and TH 6040 affect larvae of various species by interfering with cuticle development. In a biochemical study of their effects, larvae of the house fly, $\text{Musca domestica}$ L. were reared for 2 days on diets containing 1.7 to 166.7 ppm of these compounds, then assayed for activities of the microsomal oxidases and the enzyme(s) which metabolize β-ecdysone. The activities of these enzymes were compared with the percentage of treated larvae completing pupal-adult ecdysis. The two compounds reduced the activity of the β-ecdysone metabolizing enzyme(s) by as much as 57%, reduced pupal-adult ecdysis by 43% to 100%, and stimulated microsomal oxidase activity 4- to 12-fold. Supplementation of the diet of the treated insects with the Cecropia juvenile hormone, JH I, partially restored pupal-adult ecdysis but supplementation with β-ecdysone had no effect. The mode of action indicated by these results is that the IGRs cause an accumulation of β-ecdysone in the treated larvae. This stimulates the enzyme, chitinase, which degrades chitin in preparation for formation of the new cuticle. The hormone may also cause a JH deficiency and the stimulation of DOPA decarboxylase and phenol oxidase which would further disrupt the normal molting process.     

Control of ommochrome synthesis by both juvenile hormone and melanization hormone in the cabbage armyworm,Mamestra brassicae

Summary Pigmentation of last instar larvae of the cabbage armyworm,Mamestra brassicae is of two types: melanin in the cuticle and ommochrome in the epidermis. The latter was found to be primarily xanthommatin. When allatectomy was performed 8 h before head capsule slippage (HCS) in the last larval molt, later ommochrome synthesis was inhibited. Application of juvenile hormone (JH) up to 12 h after HCS (9 h before ecdysis) (activity: methopreneJH I>JH II>JH III) restored ommochrome synthesis. After that time it has less and less effect.Removal of the suboesophageal ganglion from the larvae 8 h before HCS prevented both later ommochrome synthesis and melanization. Melanization of isolated abdomens was restored by implantation of 3 suboesophageal ganglia or injection of melanization and reddish coloration hormone (MRCH) 18 h after HCS. Restoration of ommochrome synthesis required exogenous JH in addition to melanization hormone from suboesophageal ganglion or MRCH. Therefore, melanization appears to be critical for the later onset of ommochrome synthesis even in a larva which has been exposed to JH during the critical period.Abbreviations CC·CA corpora cardiaca-corpora allata complex - JH juvenile hormone - MRCH melanization and reddish coloration hormone - HCS head capsule slippage     

Critical Sensitivity of the Ovary of <Emphasis Type="Italic">Aedes aegypti</Emphasis> Adults to Sterilization by Juvenile Hormone Mimics

STERILIZATION of adult insects with juvenile hormone mimics has been reported several times^1–5, but only once for adult mosquitoes and no details were given². I report here on the sterilizing effect of three juvenile hormone mimics on female Aedes aegypti. They were (i) mixed geometric isomers of methyl 10,11-epoxy-3,7,11-trimethyl-2,6-tridecadienoate (CJH2), one of two substances with high juvenile hormone activity isolated from Cecropia oil⁶; (ii) mixed geometric isomers of farnesyl methyl ether (FME) and (iii) 2 cis/trans, 6 trans farnesenic acid ethyl ester (FAE). All three compounds were first tested on fifth instar Rhodnius prolixus using Wiggles-worth's method⁷. The doses to give a score of ten were as follows: CJH2 1.3µg; FME 1.4 µg; FAE 31.7 µg.